In the search for novel factors that play important roles in modulating clinically relevant biological processes, innovative strategies must be applied that interrogate the physical and functional interactions between biological molecules. Examples of such techniques relate to those capable of interrogating the myriad of physical interactions between multiple proteins, or in an emerging field of technology, between specific nucleic acids and proteins. Such technologies provide insight into the roles that protein-nucleic acid interactions play in modulating gene expression in the context of biological pathways. In particular, significant effort is required to identify the factors involved in controlling gene expression in disease-relevant in vivo processes, and furthermore, to identify novel means for modulating said factors.
Posttranscriptional regulation of gene expression by RNA-binding proteins (RBPs) controls a variety of cellular processes. Especially, the modulation of messenger RNA (mRNA) stability is of critical importance for the dynamic regulation of genes including transcription factors and cytokines that need to be switched on and off rapidly1,2.
The present invention has been developed through an approach interrogating protein-mRNA interactions, based on the technology PAR-CLIP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation). PAR-CLIP has been applied in identifying the mRNA molecules that physically interact with the immune regulator RC3H1 (also known as Roquin).
Roquin is an RNA-binding protein with a central role in repressing autoimmunity3. Originally, a missense mutation in the Rc3h1 gene encoding the Roquin protein was identified as the cause of systemic lupus erythematosus-like autoimmunity phenotype in sanroque mice3. Roquin binds to the 3′ untranslated region (3′ UTR) of inducible costimulator (ICOS) mRNA to posttranscriptionally repress its expression4,5. Furthermore, Roquin, as well as its paralog roquin-2, interacts with 3′UTR of TNFRSF4 and TNFalpha mRNAs, and modulates immune responses6,7. Recent studies showed that roquin protein interacts through its ROQ domain with a constitutive decay element (CDE) in the 3′ UTR of TNF mRNA and promotes the decay of this transcript by recruiting the Ccr4-Caf1-Not deadenylase complex8. The CDE of TNF folds into a characteristic stem-loop structure containing a specific trinucleotide loop, which is highly similar to the roquin RNA recognition element in the ICOS 3′UTR8. Latest structural analyses showed the ROQ domain in complex with a prototypical CDE RNA stem-loop revealing recognition of the RNA stem and its triloop9,10. Leppek and colleagues further identified additional Rc3h1 target transcripts by RIP-seq analysis, including regulators of the NF-κB pathway8. However, a recognizable CDE was absent in the majority of Rc3h1-bound mRNAs, suggesting other modes of RNA recognition8. In line with these findings Schlundt and coworkers showed by mutational and structural analyses of RNA ligands that relaxed CDE consensus sequences can mediate Roquin-dependent regulation9. In addition to the ROQ domain, RC3H1 possesses an N-terminal RING finger with a potential E3 ubiquitin-ligase function11 as well as a CCCH-type zinc finger that is potentially involved in RNA recognition. CCCH-type zinc-finger RNA-binding proteins typically contact AU-rich elements (ARE)12,13. AREs are conserved cis-regulatory elements, originally discovered in the 3′UTRs of short-lived mRNAs, encoding cytokines and early expressed immune response genes14-16.
As is described in more detail herein, RC3H1 contacts mRNAs through structure-sequence elements located in 3′UTRs. The binding sites are composed of a hairpin with embedded AU-rich, or U-rich, sequences but only to a minor extent CDE consensus sequences. RC3H1-bound mRNA targets are short-lived, and RC3H1 depletion resulted in increased protein synthesis of its target mRNAs. RC3H1 target transcripts are significantly enriched for mRNAs that are induced upon DNA damage, amongst them A20 (also known as TNFAIP3) mRNA.
The zinc finger protein A20 codes for a ubiquitin-editing enzyme, which inhibits activation of NF-κB18,19. A20 is an important negative regulator of inflammation19 and several studies have highlighted the clinical and biological importance of A20. Vande Walle and colleagues recently showed that negative regulation of the NLRP3 inflammasome by A20 protects against arthritis39.
Chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis, and type-1 diabetes, among others, are of particular importance and affect more than 50 million individuals in North America alone. Many of these diseases are debilitating and are becoming increasingly common in an aging society.
In light of the significant number of medical conditions associated with inflammation, novel means for the modulation of inflammation, in particular the reduction of inflammation associated with pathological conditions, are required in the medical field.
Antisense oligonucleotides that are capable of modulating A20 expression have been disclosed previously, for example in WO 02/20545, which discloses a number of oligonucleotides capable of inhibiting A20 expression. MicroRNA molecules have also been described that potentially bind the 3′ UTR of the A20 transcript and reduce A20 activity (Kim et al, PNAS, May 15, 2012, 109:20, 7865 and Wang et al, Biochemical and Biophysical Research Communications 411 (2011), 586). Other publications have disclosed alternative microRNA molecules that potentially protect A20 from degradation, for example by inhibiting ELAVL1/HuR binding (Balkhi et al, Sci Signal, 6(286) Jan. 9, 2014, and Zhang et al, Biochemical and Biophysical Research Communications 450 (2014), 755. None of the art in this field discloses a physical interaction between RC3H1 and A20 or whether disruption of this interaction could lead to increased A20 levels and/or activity.
The present invention therefore provides novel antisense oligonucleotides comprising a sequence targeted to the 3′ untranslated region (3′ UTR) of the TNFAIP3 (A20) transcript, capable of disrupting the RC3H1-A20 interaction, thereby increasing A20 levels and/or activity.